Multifunctional context-activated protides and methods of use

ABSTRACT

This invention is directed to multifunctional, context-activated protides that have two or more effectors with individually distinct biological functions and one or more corresponding activator sites that can each initiate or amplify the biological function of one or more effectors upon context-activation. The context-activated protides of the invention are useful in the diagnosis, prophylaxis, and therapy of a broad range of pathological conditions.

BACKGROUND OF THE INVENTION

This invention relates generally to molecular medicine and, morespecifically, to context-activated multifunctional protides.

Treatment of many diseases can be severely limited by suboptimaldistribution and/or indiscriminant toxicity of the therapeutic agent,and by resistance of the pathogenic target cells or tissue(s) to thechosen therapeutic drug. Drug resistance is a burgeoning andinternational problem of daunting concern in the treatment of infectiousdiseases, cancer, and other medical conditions.

The number of new diagnoses each year of all cancer types combinedcontinues to increase. Although cancer drugs can be effective againstmetastatic disease, their mechanism of action often leads to thesurvival of drug-resistant tumors and drug toxicity with fatalconsequences to the patient. For example, chemotherapy, while generallyan effective treatment against human cancerous diseases, is hamperedwhen the specific tumor cell-type becomes resistant to thechemotherapeutic. Overall, one of the greatest limitations on theefficacy of cancer chemotherapeutic agents is the tendency of cancercells to develop broad-spectrum resistance to a diverse panel ofanti-cancer and cytotoxic drugs. Such multiple drug resistance (MDR) isbelieved to occur to varying degrees in most cancers, either from theonset of the cancer or on recurrence following chemotherapy.

Like cancer, infectious diseases due to pathogenic bacteria, fungi,protozoa and viruses are leading causes of death worldwide. Moreover,the emergence of drug-resistant forms of these pathogens has created anurgent need for new and more effective approaches and anti-infectiveagents to combat the growing threat of microbial drug-resistance.

Microbes often become resistant to antibiotics and/or non-antibioticagents. Many conventional antibiotics retard pathogen proliferation byinteracting with and/or entering the microbes and interfering with theelaboration of microbial components or pathways needed formacromolecular metabolism (eg., proteins or nucleic acids), cellularregulation, or reproduction. For example, many conventional antibioticsfunction by impairing DNA replication or expression, transcription,ribosome function, translation, or cell wall or membrane integrity. Themajority of available anti-infective agents inhibit intracellulartargets within pathogenic microorganisms. Antibiotic resistancetypically involves individual or multiple point mutations that slightlychange the structure of the antibiotic target, for example, the cellwall synthetic enzymes or ribosomal subunit proteins, such that theantibiotic is no longer effective. Such a slight change in targetstructure with no detrimental effect on function can be sufficient toreduce or eliminate antibiotic inhibition of the target, translating toreduced or abrogated efficacy of the anti-infective agent. Other commonmechanisms for the rapid development of resistance to conventionalantibiotics include, for example, degradation of the antibiotic prior totarget inhibition, reduced permeability or access of the antibiotic toits target, and/or increased export of the antibiotic by the resistantorganism. Thus, antibiotic resistance can occur by the acquisition ofgenes encoding enzymes that inactivate agents, modify the target of theagent, or result in impermeability or active efflux of the agent.Improved methods for controlling drug resistance in microbes, inparticular, microbes that are highly drug resistant, would be oftremendous benefit.

Thus, there exists a need for therapeutic agents that circumvent or havereduced susceptibility to common mechanisms of drug resistance amongpathogenic cells, including agents of infectious disease and cancer. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

This invention is directed to multifunctional, context-activatedprotides that have two or more effectors with individually distinctbiological functions and one or more corresponding activator sites thatcan each initiate or amplify the biological function of one or moreeffectors upon context-activation. The context-activated protides of theinvention are useful in the diagnosis, prophylaxis, and therapy of abroad range of pathological conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general conceptual scheme for an invention protide.

FIG. 2 shows an extrapolated conceptual scheme for an invention protidehaving more than one activator site and more than two effectors.

FIG. 3 shows a specific example of an invention protide that isactivated in the context of vascular injury or infection.

FIG. 4 shows a further specific exemplification of an invention protidethat is activated in the context of complement fixation by C3 convertaseserine protease activators that cleave the mosaic protide at thecleavage site, liberating the effector domains IL-8 and defensin hNP-1as independent molecules to effect their respective functions.

FIG. 5 shows a general example of an invention protide that is activatedby a strategic protease activator in the context of inflammatoryresponses to tissue injury or infection trigger activation of otherwiseinactive proteases.

FIG. 6 shows (A) examples of several permutations of effector biologicalfunctions and activators useful in preparing invention protides; and (B)a table setting forth further examples of general and specific effectorsand activators useful for the preapration of an invention protide.

FIG. 7 shows the general conceptual scheme for an invention antibiotide.

FIG. 8 illustrates a specific example of an invention antibiotide.

FIG. 9 shows the general conceptual scheme for an invention antineotide.

FIG. 10 shows a specific example of an invention antineotide.

FIG. 11 demonstrates examples of several of the possible mosaiccombinations of effectors that can be incorporated into acontext-activated multifunctional protide of the invention.

FIG. 12 shows the amino acid sequence of PT-1 (SEQ ID NO: 1), PT-2 (SEQID NO: 2), PT-3 (SEQ ID NO: 3) and PT-4 (SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to multifunctional, context-activatedprotides that have two or more effectors with individually distinctbiological functions and one or more corresponding activator sites thatcan each initiate or amplify the biological function of one or moreeffectors upon context-activation. The context-activated protides of theinvention are useful in the diagnosis, prophylaxis, and therapy of abroad range of pathological conditions.

In particular, the invention protides provide novel agents andstrategies to target diagnostic, prophylactic, and/or therapeutic agentsto specific sites, to minimize cytotoxicity of otherwise toxic agents,to create relevant gradients for recruitment, potentiation, suppression,or other desirable modulation of immune system or other structural,effector or regulatory cells, and further provide many otherapplications for diagnosis, imaging, localization, prevention, ortreatment of disease, or as research tools to investigate areasincluding pathogenesis, physiology, immunobiology, cell regulation, geneexpression, or other disciplines.

The invention protides have two or more distinct biological functionsand are designed to be activated within a defined or characteristiccontext. Invention protides have the advantage of designs that can becustomized, engineered, chosen, or combined to allow for highlyselective correspondence to or association with or unique to a specificpathological condition or etiology. The distinct biological functionscan further be associated with distinct functional aims, for example,therapy, prevention, amplification and detoxification. As describedherein, a multifunctional, context-activated protide can be designed tobe activated in any context desired by the user, a feature which makesthe invention protides useful to applications in many areas of medicineand biomedical research, including, for example, diagnosis, imaging,detection, speciation or other specification, prevention/prophylaxis,and therapy of a wide range of pathological conditions such asinfectious diseases, neoplastic diseases, immune and autoimmunedisorders, cardiovascular conditions, disorders in metabolism orphysiology, diseases of inheritance or genetic abnormality, a variety ofpathological conditions associated with gene expression, mitochondrialdysfunction or regulation, as well as cell death and/or cellularsenescence.

A pathological condition appropriate for treatment with a protide can bea symptomatic disease or other abnormal condition or injury of amammalian cell or tissue. Such pathological conditions include, forexample, hyperproliferative and unregulated neoplastic cell growth,degenerative conditions, inflammatory diseases, autoimmune diseases andinfectious diseases. Hyperplastic and cancer cells proliferate in anunregulated manner, causing destruction of tissues and organs. Specificexamples of hyperplasias include benign prostatic hyperplasia andendometrial hyperplasia. Specific examples of cancer include prostate,breast, ovary, lung, uterus, brain and skin cancers.

Abnormal cellular growth can also result from infectious diseases inwhich foreign organisms cause excessive growth. For example, humanpapilloma viruses can cause abnormal growth of tissues. The growth ofcells infected by a pathogen is abnormal due to the alteration of thenormal condition of a cell resulting from the presence of a foreignorganism. Specific examples of infectious diseases include DNA and RNAviral diseases, bacterial diseases, fungal diseases, and protozoal orparasitic diseases. Similarly, the cells mediating autoimmune andinflammatory diseases are aberrantly regulated which results in, forexample, the continued proliferation and activation of immune mechanismswith the destruction of tissues and organs. Specific examples ofautoimmune diseases include, for example, rheumatoid arthritis andsystemic lupus erythmatosis. Specific examples of degenerative diseaseinclude osteoarthritis and Alzheimer's disease.

By specific mention of the above categories of pathological conditions,those skilled in the art will understand that such terms include allclasses and types of these pathological conditions. For example, theterm cancer is intended to include all known cancers, whethercharacterized as malignant, benign, soft tissue or solid tumors, orhematologic tumors relating to cells in circulation, such as leukemias.Similarly, the terms infectious diseases, degenerative diseases,autoimmune diseases and inflammatory diseases are intended to includeall classes and types of these pathological conditions. Those skilled inthe art will know the various classes and types of proliferative,infectious, autoimmune and inflammatory diseases.

As described below, in addition to their direct antimicrobialefficacies, the invention protides are useful based on their ability tocircumvent or minimize conventional resistance mechanisms by pathogensor tumor cells. For example, this can be the result of activation byactivators that are present outside of the target cell such that theprotide need not necessarily enter the target cell to be activated andto achieve subsequent efficacy, thus minimizing the likelihood forresistance due to reduced target access or increased efflux of theprotide. Furthermore, in many conventional resistance mechanisms,resistance can be induced by the presence of the anti-infective agentitself. Protides can be designed to be activated by such microbialcounter-responses or virulence factors. Thus, the more of the activatorthat is made by the organism, the more protide activation results,yielding an expected amplification of the anti-pathogenic efficacy ofthe protide. Conversely, decreased production of the activators cantranslate in turn to decreased presence or function of these sameactivators such as virulence factors or mediators of pathogenesis, inessence turning off the pathogenic potential of the target cell, orreducing its ability to protect itself from otherwise normal hostdefenses. Similarly, protides can be beneficial by reconstituting tumorcell or microbial pathogen susceptibility to conventional therapeuticagents, to which these pathogenic cells would otherwise be resistant.Thus, the invention protides can either be activated from upregulationof resistance- or virulence factor expression, or can impact efficacy byeffecting the downregulation of virulence factor expression bypathogenic cells or organisms.

The invention provides a context-activated protide encompassing theamino acid sequence set forth as SEQ ID NO: 1 and referred to herein as“PT-1,” which includes two effectors and one activator. Also provided bythe invention is a nucleic acid sequence that encodes the amino acidcorresponding to PT-1, which is set forth as SEQ ID NO: 1.

PT-1 encompasses an antimicrobial peptide effector (RP-1), achemokine-like peptide effector (IL-8 domain), and an activator sitespecific for staphylococcal V8 protease, one of numerous virulencefactors that is elaborated by S. aureus in order to establish andproliferate infection. As described in the example set forth below, PT-1is cleaved into two distinct effectors in the presence of the activator,staphylococcal V8 protease. In particular, PT-1 exerts antimicrobialactivity less than that of the antimicrobial peptide RP-1 in the absenceof V8 protease, but antimicrobial activity equivalent to or exceedingthat of RP-1 in the presence of V8 protease produced by S. aureus. Thus,PT-1 exerts optimal antimicrobial activity in the context of V8 proteaseas would be present in the setting of infections due to staphylococcalcells.

The invention also provides a context-activated protide encompassing theamino acid sequence set forth as SEQ ID NO: 2 and referred to herein as“PT-2,” which includes two effectors and one activator. Also provided bythe invention is a nucleic acid sequence that encodes the amino acidcorresponding to PT-1, which is set forth as SEQ ID NO: 2.

PT-2 encompasses an antimicrobial peptide effector (RP-1), achemokine-like peptide effector (IL-8 domain), and an activator sitespecific for C3 convertase, a complement fixing protease. As describedherein, PT-2 is cleaved into two distinct effectors in the presence ofthe activator, C3 convertase. In particular, PT-2 exerts antimicrobialactivity less than that of the antimicrobial peptide RP-1 in the absenceof C3 convertase, but antimicrobial activity equivalent to or exceedingthat of RP-1 in the presence of C3 convertase. Thus, PT-2 exerts optimalantimicrobial activity in the context of activation of one of the threecomplement pathways that make up the complement system, which is part ofthe innate immune response to antigen exposure.

The invention further provides a context-activated protide encompassingthe amino acid sequence set forth as SEQ ID NO: 3 and referred to hereinas “PT-3,” which includes two effectors and one activator. Also providedby the invention is a nucleic acid sequence that encodes the amino acidcorresponding to PT-3, which is set forth as SEQ ID NO: 3.

PT-3 encompasses an antimicrobial peptide effector (RP-1), achemokine-like peptide effector (IL-8 domain), and an activator sitespecific for thrombin, a serine-protease produced in the local contextof vascular injury or infection. As herein, PT-3 is cleaved into twodistinct effectors in the presence of the activator, thrombin. Inparticular, PT-3 exerts antimicrobial activity less than that of theantimicrobial peptide RP-1 in the absence of thrombin, but antimicrobialactivity equivalent to or exceeding that of RP-1 in the presence ofthrombin. Thus, PT-3 exerts optimal antimicrobial activity in thecontext of thrombin as would be present in the setting of vascularinjury or infection.

Also provided by the invention is a context-activated protideencompassing the amino acid sequence set forth as SEQ ID NO: 4 andreferred to herein as “PT-4,” which includes two effectors and oneactivator. Also provided by the invention is a nucleic acid sequencethat encodes the amino acid corresponding to PT-4, which is set forth asSEQ ID NO: 4.

PT-4 encompasses an antimicrobial peptide effector (RP-1), achemokine-like peptide effector (IL-8 domain), and an activator sitespecific for the matrix metalloproteinase MMP-9, which is produced todissolve the tissue in front of the growing blood vessel tip to allowfor its continued tissue invasion. As described herein, PT-4 is cleavedinto two distinct effectors in the presence of the activator, MMP-9. Inparticular, PT-4 exerts antineoplastic and/or antimicrobial activityless than that of the antineoplastic and/or antimicrobial activity ofthe peptide RP-1 in the absence of MMP-9, but antineoplastic and/orantimicrobial activity equivalent to or exceeding that of RP-1 in thepresence of MMP-9. Thus, PT-4 exerts optimal antineoplastic and/orantimicrobial activity in the context of new blood vessel formation.

In a further embodiment, the invention provides a context-activatedprotide having at least one activator site and two or more effectorswith distinct biological functions. The term “protide,” as used herein,refers to a mosaic molecule composed of two or more peptide ornon-peptide peptide functional domains, referred to as effectors, andone or more corresponding activator sites. A protide can consist of anindefinite number of effector and activator domains that can vary infunction, activation, position, continuity, or sequence. One example ofa protide described herein is PT-1 (SEQ ID NO: 1), which consists of anantimicrobial peptide effector (RP-1), a chemokine-like peptide effector(IL-8 domain), and an activator site specific for staphylococcal V8protease.

A protide of the invention can be useful in a variety of applicationsrelating to, for example, diagnosis, prophylaxis, or therapy of apathological condition. It is understood that minor modifications can bemade without destroying protide activity and that only a portion of, forexample, a particular effector or activator site can be required inorder to effect activity. Such modifications are included within themeaning of the term protide. Further, various molecules can be attachedto invention protides, including for example, conventional or newlydiscovered synthetic anti-infective agents, conventional ornewly-discovered anti-neoplastic agents, other polypeptides,carbohydrates, nucleic acids or lipids. Such modifications also areincluded within the definition of the term.

Minor modifications of a protide of the invention include, for example,conservative substitutions of naturally occurring amino acids and aswell as structural alterations which incorporate non-naturally occurringamino acids, amino acid analogs and functional mimetics. For example, aLysine (Lys) residue is considered to be a conservative substitution forthe amino acid Arg. A protide containing one or more mimetic structureshaving a similar charge and spatial or steric arrangements as thereference amino acid residues is included within the definition of theterm so long as the protide containing the mimetic portion exhibits asimilar or enhanced activity as compared with the reference protide. Itis thus understood that an invention protide includes such mimetics aschemically modified peptides, peptide-like molecules containingnon-naturally occurring amino acids, and peptoids, which arepeptide-like molecules resulting from oligomeric assembly ofN-substituted glycines, with similar or enhanced activity as comparedwith the reference protide upon which the mimetic is derived or havingany other property desired by the user, for example, enhancedbiostability (see, for example, Goodman and Ro, Peptidomimetics for DrugDesign, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed.M. E. Wolff; John Wiley & Sons 1995), pages 803-861), which isincorporated herein by reference in its entirety.

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules that contain a constrained amino acid, anon-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; an α,α-dialkyl-glycine or α-aminocycloalkanecarboxylic acid; an Nα-Cα cylized amino acid; an Nα-methylated aminoacid; a β- or γ-amino cycloalkane carboxylic acid; an α,β-unsaturatedamino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an N-Cδ or Cα-Cδ cyclized aminoacid; or a substituted proline or another amino acid mimetic. Inaddition, a peptidomimetic that mimics peptide secondary structure cancontain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylenesulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that an invention protidecan encompass these and other peptidomimetics. Likewise, an inventionprotide also can contain stereoisomeric amino acids or other effector oractivator constituents, such as dextrorotatory (D) versions of aminoacids.

As described herein, a protide can contain naturally occurring andnon-naturally occurring amino acids as well as amino acid analogs andmimetics. Naturally occurring amino acids include the 20levorotatory(L)-amino acids utilized during protein biosynthesis as wellas others such as 4-hydroxyproline, hydroxylysine, desmosine,isodesmosine, homocysteine, citrulline and ornithine, for example.Non-naturally occurring amino acids include, for example, (D)-aminoacids, norleucine, norvaline, p-fluorophenylalanine, ethionine and thelike. Amino acid analogs include modified forms of naturally andnon-naturally occurring amino acids. Such modifications can include, forexample, substitution or replacement of chemical groups and moieties onthe amino acid or by derivitization of the amino acid. Amino acidmimetics include, for example, organic structures which exhibitfunctionally similar properties such as charge and charge spacingcharacteristic of the reference amino acid. For example, an organicstructure which mimics Arginine (Arg or R) would have a positive chargemoiety located in similar molecular space and having the same degree ofmobility as the α-amino group of the side chain of the naturallyoccurring Arg amino acid. Mimetics also include constrained structuresso as to maintain optimal spacing and charge interactions of the aminoacid or of the amino acid functional groups. Those skilled in the artknow or can determine what structures constitute functionally equivalentamino acid analogs and amino acid mimetics useful for preparation of aninvention protide.

Specific examples of amino acid analogs and mimetics can be founddescribed in, for example, Roberts and Vellaccio, The Peptides:Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341,Academic Press, Inc., New York, N.Y. (1983), the entire volume of whichis incorporated herein by reference. Other examples include peralkylatedamino acids, particularly permethylated amino acids. See, for example,Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, JohnWiley & Sons Inc., New York, N.Y. (1997), which is incorporated hereinby reference in its entirety. Yet other examples include amino acidswhose amide portion and, therefore, the amide backbone of the resultingpeptide, has been replaced, for example, by a sugar ring, steroid,benzodiazepine or carbo cycle. See, for example, Burger's MedicinalChemistry and Drug Discovery, supra, Ch. 15, pp. 619-620, which isincorporated herein by reference in its entirety. Methods forsynthesizing peptides, polypeptides, peptidomimetics and proteins arewell known in the art (see, for example, U.S. Pat. No. 5,420,109; M.Bodanzsky, Principles of Peptide Synthesis (1st ed. & 2d rev. ed.),Springer-Verlag, New York, N.Y. (1984 & 1993), see Chapter 7; Stewartand Young, Solid Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co.,Rockford, Ill. (1984), each of which is incorporated herein by referencein its entirety).

The term “context-activated,” as used herein in reference to a protideof the invention, refers to the initiation, activation or amplificationof a biological or other desired, for example, diagnostic orprophylactic function of one or more protide effectors in a particulartemporal, spatial, pathological and/or biochemical context.Context-activation can be initiated by direct or indirect interactionbetween a protide activator site and a corresponding activator that isselectively associated with the particular context. As used herein,context-activation encompasses activation in a wide variety of contextsthat can include, for example, local, regional, systemic, and/ortemporal proximity; as well as the presence or absence of an etiologicalagent, pathologic condition, or characteristic components thereof.

Thus, context need not be limited to a place, time or quality, but alsocan be the presence or absence of an activator, for example, an enzymeelaborated by an organism such as, for example, a specific strain ofbacteria. The context for activation can consequently be of any breadthdesired by the user, for example, can target a class of organisms orcell types, for example, by using an activator that is ubiquitous to thetargeted class, or can alternatively have a more narrow focus by usingan activator that represents a more narrowly defined target, forexample, a particular genus, organism, species, subspecies, strain, orcell or tissue type. The context can be associated with a pathologicalcondition, but also can be selected to represent a non-pathologicalenvironment, for example, in prophylactic applications of the inventionpracticed to preserve a normal or homeostatic condition.

As used herein, the term “effector” refers to the peptide or non-peptidefunctional domains of an invention protide that have specific individualfunctions, which are initiated or amplified upon activation and achievespecific functions relating to the diagnosis, prevention, or treatmentof a disease. As described herein, an invention protide has at least twoeffector domains with distinct, complementary and/or synergisticbiological functions. An effector is inactive or exhibits relativelyreduced or attenuated biological activity unless an activator, by virtueof either its presence or absence, alters or facilitates or allows thealtering of its corresponding activator site and, as a result, initiatesor amplifies the diagnostic, prophylactic, therapeutic, or otherbiological function(s) of the effector(s). Multiple effectors can beinduced by the same activator site. Peptide and non-peptide effectorscan be present in the same protide, which can be referred to as a hybridprotide. Similarly, a protide can consist exclusively of peptideeffectors, also referred to as a peptide protide. Similarly, a protideof the invention can consist exclusively of non-peptidic effectors. Thebiological function(s) of an effector that corresponds to an inventionprotide can be, for example, antimicrobial, immunomodulatory, pro- oranti-inflammatory, tumoricidal, pro- or anti-apoptotic, pro- andanti-angiogenic and/or hemolytic.

As described herein, a protide of the invention can be bifunctional ormultifunctional, with two or more unique complementary effectors, andone or more activators as determined by specific effector and activatorsite domains engineered into the mosaic protide, which can be activatedby specific molecules or conditions present in unique or strategiccontexts of interest. Examples of such effectors can include one or moreantimicrobial, anti-neoplastic, anti-inflammatory, immunomodulatory, orother peptide or non-peptide functional domains, or combinationsthereof.

As used herein, the term “activator site” when used in reference to aprotide of the invention, refers to a domain of the protide that, in thepresence of an activator, initiates, promotes, amplifies or modulatesthe specific biological function of one or more effectors. As describedherein, an activator site can be modified, cleaved, processed orotherwise altered in the presence of an activator. In addition, anactivator site can be sensitive either to the absolute presence orabsence of an activator as well as can be sensitive to a thresholdconcentration of an activator rather than its mere presence.

An activator site useful in the invention can include one or more sitesfor cleavage, modification, processing or other triggering by strategicactivators, which can be, for example, proteases, esterases, lipases, orother endogenous enzymatic activators or cascades generated by orassociated with a specific condition such as, for example, the presenceof pathogenic microorganisms, damaged or inflamed tissues, orhematologic or solid neoplastic or pre-neoplastic cells or tumors. Suchan activator site also can be selected to exploit contexts associatedwith biochemical or physical conditions such as requisite acidity oralkalinity, for example, acidic phagolysomes containing intracellularbacteria or fungi; or ionic or osmotic strength, for example, in a renalcontext, that represent a specific pathologic or non-pathologic context.Furthermore, an activator site can be selected to exploit normal ratherthan a pathologic context.

An activator site can be subject to proteolytic as well asnon-proteolytic activation. For example, the activator site can belocated within the peptide moiety, and require a protease activator. Inother embodiments, the non-proteolytic activator can target anon-proteinaceous substrate component of the protide. For example, aprotide of the invention can include an esterase activator and can linkpeptide and/or non-peptide moieties (eg. a protide consisting of peptideand conventional antibiotic effectors) by means of an ester bond. Otherbiochemically relevant bonds or linkages that can serve as activationsites in an invention protide can include, for example, lipase- (lipidcleaving), nuclease- (nucleic acid cleaving), and kinase or phosphatase-(phosphate addition or removal) sensitive activators that targetsubstrates other than peptides. For example, certain microbial pathogensor tumor cells can express, or abnormally express restriction enzymesthat can provide a suitable basis for design of a protide that could beactivated only within the target cell, further reducing indiscriminanthost cytotoxicity.

As used herein, the term “activator” refers to a molecule or conditionthat, by altering the activator site, causes the liberation or onset ofa specific diagnostic or biological function of effector(s). Asdescribed herein, an activator can be a normal or abnormal exogenous orendogenous cell, structure or molecule, a condition or milieu (normal orabnormal), or a combination thereof that is associated with a specificcontext in which activation of the protide is desired. Thus, anactivator can be selected based on its presence in a temporal, spatial,or physiological context, which can be normal or abnormal, that isassociated with the desired context for protide activation. An activatorcan consequently include physiological conditions including, forexample, acidity, alkalinity, conditions of oxidation or reduction,and/or ionic and/or osmotic strength, that are associated with aparticular context, and modulate protide activation. Alternatively, anactivator can be a structure or molecule, for example, an enzyme, thatis present in a particular spatial, temporal or pathological context.The activator molecule can modify the activator site upon association,for example, by cleavage or other modification that results inactivation in the particular context, or can facilitate interactionbetween protide and activator(s). The activator molecule can be anenzyme including, for example, protease, esterase, lipase, nucleases orpeptidase.

In one embodiment of the invention, an activator site can encompass oneor more domains for cleavage, modification, processing or any other typeof liberation by an activator, for example, a protease, esterase, lipaseor other endogenous or exogenous enzymatic activator or cascade. Thechoice of one or more activator sites that correspond to specificactivators depends directly on the desired context for activation. Thus,an activator can be a particular pathologic setting or condition that ischosen based on its association with a particular etiological agent orhost response. In the presence of the activator, one or more effectorsare liberated so as to achieve a specific function relating to, forexample, the treatment, prevention, or diagnosis of a targeted disease.An activator site can thus be strategically designed to become activatedin temporal and spatial proximity to activator expression, therebyallowing the activation of a protide to be targeted to a particularcontext and over time so as to maximize the desired therapeutic orprophylactic effect, while minimizing untoward or undesirable toxicitiesor other side effects.

As described herein, an activator site is selected based on itscorrespondence and/or association with the context in which the two ormore protide effectors are to be liberated so as to initiate orpotentiate their functions. Therefore, as long as an activator isassociated with the context, the invention can be practiced with anycontext desired. Those skilled in the art will appreciate that, giventhe versatility of activators useful for practicing the invention asdescribed herein, a protide can be designed based on virtually anycontext desired, inluding, for example, vascular injury, presence of aneoplasm or cancer, infection, and inflammation.

In one embodiment, the protide is an antimicrobial protide, which alsocan be referred to as an antimicrotide. Cleavage sites for strategicproteases can be engineered into multifunctional antimicrobial protidesso as to represent the activator site of the protide. Upon activation ofthe protease in the localized or generalized context of tissue injury orinfection, as selected by the user, the inactive protide is cleaved,liberating independent and active molecules to effect their respectivebiological functions. Prior to and beyond the setting of activation ofthe strategic protease representing the activator, the mosaic protideconstruct is relatively inactive both with respect to antimicrobialfunction and host cell toxicity. A mosaic protide construct can consistof an indefinite number (1 through n) of effector and activator domainsthat can vary in function, activation, position, continuity, orsequence. Effectors corresponding to one or more protides activated bythe same or distinct activators also can function synergistically,and/or can recombine in a manner facilitating their complementaryfunctions.

As an example, in the context of vascular injury, a protide activatorcan be selected that specifically represents this particular context,for example, a clotting cascade protease such as thrombin, or acomplement fixing protease such as a C3 convertase, for example, C4B2Aor C3bBb. Similarly, as another example, a protide activator can beselected that represents a broader constellation of symptoms orconditions, such as sepsis, in which corresponding activators caninclude serine proteases associated with systemic inflammation, sepsis,or injury, such as activated protein C.

A further embodiment of the invention encompasses anti-neoplasticprotides, which also are referred to as antineotides. Many tumor cellsproduce or overexpress characteristic activators, such as matrixmetalloproteinases (MMP) or other enzymes that are not expressed by, orat levels much higher than normal cells. Consequently, the activator canbe a tumor-specific protease, for example, a matrix metalloproteinase orthymidylate synthase (TS), which is overexpressed in the majority ofcancers. A tumor-specific protease also can be associated with a morenarrow neoplastic context, such as a serine protease that isspecifically expressed in prostate cells, for example, PSA, humankallikrein-2 (hK2), human kallikrein-11 (hK11) and TMPRSS2.

In the example shown in FIG. 10, metalloproteinase can serve as anactivator that cleaves the mosaic antineotide at the activator site. Inthis example, one effector domain is an anti-angiogenic peptide thatwould serve to restrict vascular access to a tumor, the other is a moreconventional antineoplastic chemotherapeutic agent. Each domain isliberated in the context of metalloproteinase or other relevantactivator as independent molecules to effect their individual and/orcomplementary antineoplastic functions. Prior to and beyond the localsetting of activation, the mosaic construct can be significantly lesstoxic to normal cells than either individual domain. Thus, contextactivation at sites of tumor cell activity localizes and activates theantineotide.

In yet another embodiment, the context can be the presence of aninflammatory response as described above, and the effector(s) can becytokine functional groups, such as those of interleukin-1 or tumornecrosis factor-alpha, or chemotactic cytokines (chemokines) such asinterleukin-8, IP-10 or MIG, known to be involved in the coordination,for example, trafficking or navigation, of T-lymphocytes, neutrophils,macrophages, or other immune effector or immunoregulatory cell types tothe site of inflammation. The nonapeptide bradykinin is a classicmediator of inflammatory response that is generated from high molecularweight precursors termed kininogens by limited proteolysis mainly inresponse to tissue injury. Most of the biological actions of bradykininare mediated through at least two different receptors, the bradykinin B1and B2 receptors.

The proteins encoded by the serine protease gene family are usefulactivators in a variety of contexts. Serine proteases areprotein-cleaving enzymes that play important roles in normal andpathological physiological processes including protein processing ordigestion, for example, trypsin and chymotrypsin; tissue remodeling, forexample, stratum corneum chymotryptic enzyme and urokinase; bloodcoagulation; for example, plasminogen activator and thrombin; fertility,for example, acrosin; inflammatory responses, for example, elastase;tumor cell invasion, for example, uPA3; and programmed cell death, forexample, granzymes. Thus, serine proteases are associated with a varietyof contexts and can be selected as activators for an invention protidetargeted for activation in any of the contexts with which they areassociated. It is understood that, depending on the desired application,a specific protide activator site can be any enzyme, including, forexample, a lipase, esterase, kinase, endo- and exonuclease.

One example of an activator molecule is a protease expressed by abacterial pathogen, such as a sortase enzyme expressed by Staphylococcusaureus (S. aureus). The bacterial pathogen S. aureus is increasinglyresistant to most conventional antibiotics, including, for example,methicillin and vancomycin, and is associated with increasing mortalityand morbidity. S. aureus produces several virulence factors that arenecessary to achieve and propagate infection, among them sortase, atranspeptidase that is essential for S. aureus to attach to its surfacemolecules that are necessary for binding, immunoavoidance, andvirulence, promoting the ability of this organism to cause infection. Aprotide can be prepared integrating an activator site that is aspecifically recognized substrate for sortase as the activator. In thecontext of an S. aureus infection, sortase will naturally be expressedand serve to activate the protide by cleavage of the activator site.Depending on the effectors, which can have for example, distinctantimicrobial and immunomodulatory functions that are activated uponcleavage of the activator site, the bacteria will be killed by theantimicrobial effector, while immune effector cells will be recruited tothe site by the immunomodulatory effector. In this example, if theorganism responds by compensatory increases in sortase expression, moreprotides will be activated. Alternatively, if the organism ceasessortase expression, it no longer will be able to attach necessaryproteins to its surface and, consequently, will have reduced or novirulence. Moreover, since sortase is present on the extracellularaspect of the organism, protides do not accumulate within the targetcell, likely circumventing efflux-mediated resistance. Furthermore, aninvention protide can contain distinct effectors that, upon activation,can inhibit one or more sortase type enzymes, and target anotheressential structure such as the staphylococcal membrane. It isunderstood that such other enzymes known in the art to mediate virulencein both Gram positive and Gram negative bacteria also are examples ofuseful activators of protides designed to have a broad antibacterialspectrum. Alternatively, more narrowly defined protides can be designedwith activator(s) present only in the context of one or a few specifictypes of bacteria. Moreover, based on the teachings provided herein, theskilled person can design and prepare analogous protide effectorconstructs and corresponding activator target sets in other pathogens,including other drug resistant bacterial species, as well as importantfungal pathogens such as Candida albicans, and viral pathogens such ashuman hepatitis C virus and HIV.

Thus, activation of a protide of the invention takes place in a contextassociated with or unique to a specific condition. In addition or as analternative to inhibiting a target or process directly, a therapeuticstrategy highly susceptible to mutation-based resistance, a protide ofthe invention can be specifically designed to, for example, subvertessential virulence factors or pathogenic mechanisms as a means todiagnose, prevent, or treat, disease. Most conventional antibioticsretard viral, bacterial, fungal, or protozoal proliferation by enteringthe microbes and interfering with the production of components needed tofor homeostasis, metabolism, synthesis, or reproduction, for example, byimpairing DNA, RNA, or protein manufacture, or perturbing cell wall ormembrane structures. Antibiotic resistance typically involves one ormore simple point mutations that slightly change the structure ofantibiotic target, for example, the cell wall or ribosomal subunits,such that the antibiotic is no longer effective. Such a slight change instructure with no effect on function is sufficient to inhibit theantibiotic's effect upon the target. Another common reason for rapidlydeveloping resistance against conventional antibiotics is that an enzymethe pathogen naturally produces interferes with the antibiotic such theantibiotic does not reach its target structure in an active form or insufficient concentration to be efficacious.

An antimicrobial protide of the invention consisting solely of peptideeffectors (also referred to an antimicrotide), can include two or moreeffectors that encompass, for example, an antimicrobial peptide,toxicity-neutralizing peptide, immunomodulatory peptide,ligand-targeting peptide, or any other polypeptide sequence expected tohave specific function(s) when activated in the context of microbialinfection or host cellular, tissue, organ, or systemic response totissue injured due to infection. Antimicrobial peptides that exertpotent microbicidal action against pathogens, including those that areresistant to conventional antibiotics, are useful as an effector in aprotide of the invention. Antimicrobial peptides can be toxic to humanor mammalian cells if indiscriminately targeted, or can have suboptimalactivities in complex biomatrices such as blood, plasma, or serum due toinactivation or inadequate accumulation at sites of infection. Theseundesirable properties can be avoided by utilizing antimicrobialpeptides as effectors in the invention protides, where they remainrelatively inactive until specifically activated either by activatorssuch as the microbial target cells or their components in the context ofinfection, by activators generated by tissue injured due to infection,or combinations thereof.

Antibiotides illustrate another subset of anti-infective protides andrepresent useful protides of the invention based on their ability toprevent or treat infection, and/or subvert bacterial drug resistancemechanisms. Rather than inhibiting a bacterial target or processdirectly as attempted by conventional antibiotics, a therapeuticstrategy that is highly susceptible to mutation-based resistance,antibiotides are designed to subvert essential virulence factors orpathogenic mechanisms to prevent or treat disease.

FIG. 7 shows the general conceptual scheme for an invention antibiotide.An example of an invention antibiotide consists of an activator sitethat is cleaved by the activator thrombin, liberating two effectors, theantimicrobial peptide (AP) and beta-lactam (b-lactam) antibiotic (FIG.8). Thrombin is a serine-protease produced in the local context ofvascular injury or infection. In this context, thrombin is present andcleaves the antibiotide activator site leading to activation of theeffectors, the antimicrobial peptide (AP) and beta-lactam antibiotic,which have individual and synergistic antimicrobial functions. Inparticular, the AP represents a potent microbicidal peptide, while theb-lactam antibiotic exert a distinct, but complementary antimicrobialeffect. It is understood that conventional antibiotics other thanb-lactam class agents such as, for example, quinolones,anti-metabolites, aminoglycosides, rifampins and rifamycins,tetracyclines, macrolides, azoles, and any other class of therapeutic orprophylactic agent, anti-infective or other, also are useful effectorsin an invention antibiotide. Prior to thrombin-activation, the mosaicprotide construct is relatively inactive, minimizing or reducing thenon-selective toxicity of AP, while the b-lactam is protected fromb-lactamase enzymes which can degrade the antibiotic component. Thus,presence of the activator thrombin in the context of vascular infectionlocalizes and activates the prodrug antibiotide. Moreover, activation ofa protide containing an effector that increases target cell permeabilityto another effector such as, for example, to a conventional antibiotic,can significantly augment the efficacy of one or both effector species,or of the combination of such, by allowing increased access tointracellular targets, or circumvent efflux or impermeability resistancemechanisms.

As described herein, context-activated protides offer multiple potentialadvantages as compared with conventional diagnostic, prophylactic, ortherapeutic agents, including sensitivity to an increase in expressionof an activator, for example, a microbial virulence factor or hallmarkindicator, which results in a corresponding direct increase in protideactivation and function. Significantly, context-activated protides canallow for, favor and promote survival of microbial, non-malignant, orother cells that do not express virulence factors or other unfavorabletraits, which would otherwise activate protides resulting in injuriousor lethal consequences to the activating target cell. Thus, a protide ofthe invention can be prepared that does not indiscriminately inhibit orkill, for example, normal flora microorganisms, that can often provideadditional contributions to host defense and health. As describedherein, a protide of the invention has characteristics that can alsofavor suppression of expression or hyper-expression of virulencefactors, which often accompanies treatment with conventional antibioticsof microbial or other target cells, for example, bacteria thathyper-express toxins as they are being killed by classical antibiotics.Thus, use of invention protides avoids the increased expression oftoxins that is associated with conventional therapies and that can leadto worsened or accelerated morbidity and mortality. Significantly, acontext-activated invention avoids promoting the induction of classicalor conventional resistance mechanisms by being designed to target thesecells, their resistance mechanisms, or the molecules or pathways thatconfer these mechanisms as the intended activator(s).

Antineotides represent a further subset of the invention protides. FIG.9 shows a general conceptual depiction of an invention antineotide. Aninvention antineotide can be designed to contain one or more activatorsites that are responsive to tumor cell-characteristic activators, suchas matrix metalloproteinases, which are enzymes that are either notexpressed or expressed at much lower levels in normal cells, allowingthese enzymes to be exploited as activators that cleave the mosaicantineotide at the activator site.

It is understood that additional activators generally or specificallycorresponding to neoplastic target cells can also be useful componentsof an antineotide mosaic protide of the invention. For example, anantineotide can be composed of a permeability increasing effector suchas a peptide, and an antisense nucleic acid sequence, such thatactivation of the protide into its peptide and nucleic acid componentsprovides a highly specific and advantageous means of inhibition orkilling of the target cell. As a consequence of permeabilization of thetarget cell by the peptide, sufficient levels of the antisense nucleicacid sequence can enter the cell, hybridize with and block thecorresponding complementary strand of nucleic acid, thus inhibitingcrucial functions such as DNA replication or mRNA expression,transcription, and/or translation. Thus, an invention protide can bedesigned that, upon context activation, introduces a nucleic acid into acell.

In the example of an antineotide mosaic protide shown in FIG. 10; oneeffector domain is an antiangiogenic peptide, the other is anantineoplastic chemotherapeutic agent. Each domain is liberated in thecontext of metalloproteinase or other relevant activator as independentmolecules to effect their individual and synergistic antineoplasticfunctions. Moreover, prior to activation, the mosaic construct can besignificantly less toxic to normal cells than either individual domain.Thus, context activation at sites of tumor cell activity localizes andactivates the antineotide.

Those skilled in the art will appreciate that many variations of theprotides exemplified herein can be prepared based on many strategic orfavorable permutations encompassing the activator sites as well as onthe effectors that are chosen based on the particular context activationand biological functions, respectively, desired by the user. A protideof the invention can consist of two or more effectors having at leasttwo distinct diagnostic or biological functions. The effectors containor are separated by one or more strategic activator sites. For example,variations, of the above protide example can contain distinct effectorsthat, upon activation, inhibit one or more sortase enzymes directly, andtarget another essential structure such as the staphylococcal membrane,or an intracellular target or pathway. In addition to these strategies,protides are designed to be relatively inactive at sites other thantheir appropriate microenvironmental context such that inadvertenttoxicity or activation is minimized. Given the teachings providedherein, it is understood that similar paradigms can be extended to abroad range of potential therapeutic applications for protides of theinvention.

Set forth in FIG. 6, are examples of several permutations of effectorbiological functions and activators useful in preparing an inventionprotide. Those skilled in the art will appreciate that the combinationsset forth herein are only exemplifications of the invention protideconcept and that the user can translate the protide concept into anydesired combination of effectors and activator sites so as to customizeand invention protide having desired biological functions that areactivated in a specific context. It is understood that a given protidecan be narrow or broad in spectrum, strategically corresponding to thedegree to which activator expression is conserved among or unique topotential target cells or tissues. Set forth in FIG. 6(B) are examplesof general and specific effectors and activators useful for thepreparation of an invention protide.

FIG. 11 illustrates examples of both general and specific activators andeffectors. As described herein, based on the teachings provided by thisinvention those skilled in the art will be able to design or customize aprotide or combination of protides that encompasses any permutation ofthe activators and effectors described herein as well as others known inthe art. Mosaic combinations of context-activated multifunctionalprotides can encompass, for example, anti-infective, anti-neoplastic,immune modulating and cascade regulating functions.

A protide of the invention employs at least two effectors individuallyhaving distinct biological functions, for example, antimicrobial andimmunomodulating; antimicrobial peptide and complementary antibiotic; orenzyme-inhibitor and antimicrobial peptide. Effectors can includepolypeptides and non-peptidic molecules, including, for example,conventional antibiotic compounds, antineoplastic compounds,immunomodulatory compounds, apoptotis-inducing compounds,apoptosis-inhibiting compounds, and apoptosis-modulating compounds.Consequently, a protide of the invention can contain combinations orsets of any two or more biological functions, for example, anti-toxicand antimicrobial, anti-neoplastic and anti-angiogenic, antimicrobialand immunomodulatory, or pro-apoptotic and anti-angiogenic.

If desired, a protide of the invention can be designed so as to deriveits own context. In particular, an effector, for example, anantimicrobial peptide, can be chosen that seeks out or accumulates inthe presence of the target until a critical or threshold concentrationis achieved in a localized area. In this regard, antimicrobial orantineoplastic protides can be further specified as to their context foractivation. Likewise, strategic or diagnostic protides withimaging-detectable labels are useful to localize the specific area(s) ofdisease process; in the absence of disease the protides are clearedwithout rendering a definitive or characteristic signal. In certainembodiments of the invention, an effector also can be selected toself-amplify or self-perpetuate, or to catalyze or modulate endogenousregulatory cascades. As a result, a protide of the invention also can bedesigned to amplify a process, reaction or cascade that occurs naturallyeven in the absence of the protide. As described herein, acascade-regulating protide of the invention, also referred to ascascatide, can promote, suppress, or otherwise modulate biologicalpathways, for example, signal transduction, coagulation, apoptosis.

As described herein, an effector having an antibiotic biologicalfunction can act directly by inhibiting a virulence factor, such assortase, that acts as an activator, or can act indirectly by targetinganother essential structure, for example, the bacterial membrane.

An effector with an immunomudulatory function can consist of animmunomodulatory molecule that, upon activation of the effector,mediates an immune response that is specific for a target antigen ormechanism of pathogenesis, or it can be nonspecific. A specificimmunomodulatory molecule alters an immune response to a particulartarget antigen. Examples of specific immunomodulatory molecules includemonoclonal antibodies, including native monoclonal antibodies, drug-,toxin- or radioactive compound-conjugated monoclonal antibodies, andantibody-dependent cell cytotoxicity-(ADCC) or phagocytosis-targeting(eg., opsonizing) molecules. Such immunomodulatory molecules stimulatean immune response by binding to antigens and targeting opsonized cellsfor inactivation or destruction. An effector with an immunomudulatoryfunction can consist of an immunomodulatory molecule to suppress animmune response to an antigen. For example, a toleragenizing moleculecan be used to suppress an immune response to a self-antigen.

An effector with an immunomodulatory function also can consist of anon-specific immunomodulatory molecule that can stimulate or inhibit theimmune system in a general manner through various mechanisms that caninclude, for example, stimulating or suppressing cellular activities ofimmune system cells. Nonspecific immunomodulatory molecules useful forstimulating an immune responses include, for example, agents thatstimulate immune cell proliferation, and/or immune cell activation andproduction of cytokines and co-stimulatory molecules. Well knownimmunomodulatory molecules that modulate immune response are, forexample, interleukins, interferons, levamisole and keyhole limpethemocyanin. Nonspecific immunomodulatory molecules useful forsuppressing immune responses include, for example, agents that inhibitcytokine synthesis or processing, specific cytokine receptor blockingreagents such as soluble receptors and receptor antagonists, andcytokines that down-regulate or inhibit the production of otherimmunomodulatory molecules. Well known immunomodulatory molecules forsuppressing an immune response include, for example, cyclosporin,rapamycin, tacrolimus, azathioprine, cyclophosphamide and methotrexate.Immunomodulatory molecules can be contained in a mixture of molecules,including a natural or man-made composition of molecules. Exemplarynatural compositions of immunomodulatory compounds include, for example,those contained in an organism such as Bacille Calmette-Guerin orCorynbacterium parvum. Exemplary man-made immunomodulatory compositionsof molecules include, for example, QS-21, DETOX and incomplete Freund'sadjuvant.

As described above, the biological function of an effector thatcorresponds to an invention protide can be antimicrobial. An effectorwith an antimicrobial biological function can consist of anantimicrobial molecule that, upon activation of the effector, mediatesan antimicrobial response.

Antimicrobial peptides generally are broad spectrum anti-microbialagents that can be useful as effectors in an invention protide. Thesepeptides, which are useful as effectors in certain invention protides,typically disrupt microbial cell membranes and other essential processesor targets, leading to eventual cell death that may or may not involvecell lysis. Over 500 antimicrobial peptides occur naturally. Inaddition, analogs have been synthesized de novo as described inJavadpour et al., J. Med. Chem. 39:3107-3113 (1996); and Blondelle andHoughten, Biochem. 31: 12688-12694 (1992), each of which is incorporatedherein by reference. A special group of membrane interacting antibioticsare pore forming peptides like alamethicin, gramicidin A, and melittin,which kill cells by perforating the electrochemical gradients ofmembranes and depleting their energy storage. While some antimicrobialpeptides such as melittin are non- or poorly selective and damage normalmammalian cells at the minimum microbicidal concentration, others aremore selectively toxic for specific microbial pathogens, such asbacterial cells. For example, the naturally occurring magainins andcecropins exhibit substantial bactericidal activity at concentrationsthat are not typically lethal to normal mammalian cells. These and otherknown antimicrobial peptides can be useful as effectors in an inventionprotide.

An antimicrobial peptide useful as an effector in an invention protidefrequently contains cationic amino acids, which are attracted to thehead groups of anionic phospholipids, leading to the preferentialdisruption of negatively charged membranes. Once electrostaticallybound, the amphipathic helices can distort the lipid matrix, resultingin loss of membrane barrier function (Epand, The Amphipathic Helix, CRCPress: Boca Raton (1993); Lugtenberg and van Alphen, Biochim. Biophys.Acta 737:51-115 (1983), each of which is incorporated herein byreference). Prokaryotic cytoplasmic membranes maintain largetransmembrane potentials and have a high content of anionicphospholipids. In contrast, the outer leaflet of eukaryotic plasmamembranes generally has low membrane potential and is almost exclusivelycomposed of zwitterionic phospholipids. Thus, due to such distinctionsin membrane compositions and/or energetics, antimicrobial peptides canpreferentially disrupt prokaryotic membranes as compared to eukaryoticmembranes and are useful as effectors in a protide of the invention.

An effector having antimicrobial biological function that is initiatedor amplified upon activation can consist of a naturally occurring orsynthetic peptide having antimicrobial activity, which is the ability tokill or slow the growth of one or more microbes. An antimicrobialeffector can, for example, kill or slow the growth of one or morestrains of bacteria including a Gram-positive or Gram-negative bacteria,as well as fungi or protozoa. Thus, an antimicrobial effector can have,for example, bacteriostatic or bacteriocidal activity against, forexample, one or more strains of Escherichia coli, Pseudomonas aeruginosaor Staphylococcus aureus. While not wishing to be bound by thefollowing, an antimicrobial effector can have biological activity due tothe ability to form ion channels through membrane bilayers as aconsequence of self-aggregation.

An antimicrobial peptide useful as an effector in an invention protideis typically basic and can have a linear or cyclic structure. Asdiscussed further below, an antimicrobial peptide can have anamphipathic alpha-helical structure (see U.S. Pat. No. 5,789,542;Javadpour et al., supra, 1996; Blondelle and Houghten, supra, 1992). Anantimicrobial peptide useful as an effector in an invention protide alsocan be, for example, a beta-strand/sheet-forming peptide as described inMancheno et al., J. Peptide Res. 51:142-148 (1998).

An antimicrobial peptide useful as an effector in an invention protidecan be a naturally occurring or synthetic peptide. Naturally occurringantimicrobial peptides have been isolated from biological sources suchas bacteria, insects, amphibians and mammals and are thought torepresent inducible defense proteins that can protect the host organismfrom bacterial infection. Naturally occurring antimicrobial peptidesinclude the gramicidins, magainins, cryptdins, defensins and cecropins(see, for example, Maloy and Kari, Biopolymers 37:105-122 (1995);Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994); Bessalle et al.,FEBS 274:151-155 (1990); and Blondelle and Houghten in Bristol (Ed.),Annual Reports in Medicinal Chemistry pages 159-168 Academic Press, SanDiego, each of which is herein incorporated by reference). Anantimicrobial peptide also can be an analog of a natural peptide,especially one that retains or enhances antimicrobial potency orselectivity.

An antimicrobial peptide incorporated within a protide of the inventioncan have low mammalian cell toxicity as can readily be determined viaroutine assays. For example, mammalian cell toxicity can be estimated bylysis of human erythrocytes in vitro as described in Javadpour et al.,supra, 1996. An antimicrobial peptide having low mammalian cell toxicitycan be less lytic to human erythrocytes, or require concentrationssignificantly exceeding those required for antimicrobial activity, ascompared with highly toxic peptides. It is understood, that anantimicrobial peptide incorporated within a protide of the inventionalso can have considerable mammalian cell toxicity that can be modulatedby the context activation.

A microbe is a minute life form that can include, for example, bothprokaryotic and eukaryotic organisms, as well unicellular andmulticellular organisms. Bacteria are a specific example of unicellular,prokaryotic microbes whereas protozoa such as amoebas, ciliates,flagellates, and sporozoans are specific examples of unicellulareukaryotic microorganisms. Examples of multicellular eukaryoticmicroorganisms include, for example, multicelluar fungi such as molds.Microbes can vary in many ways, such as morphology, virulence factorexpression, composition and structure, oxygen and nutritionalrequirements, motility, susceptibility or resistance to antimicrobialagents, and many other aspects.

As described above, the biological function of an effector incorporatedwithin a protide of the invention can also be antineoplastic ortumoricidal, terms used interchangeably herein. Furthermore, thebiological function of an effector incorporated within a protide of theinvention can be pro- or anti-apoptotic. In addition, the biologicalfunction of an effector incorporated within a protide of the inventioncan be pro- or anti-angiogenic.

A neoplastic cell is a cell that proliferates without normal homeostaticgrowth control resulting in a benign or malignant lesion ofproliferating cells. Such a lesion can be located, for example, in thegastrointestinal system, such as in the colon, small intestine, stomach,appendix and rectum. Cancer describes a class of diseases characterizedby the uncontrolled growth of aberrant cells, including all knowncancers, and neoplastic conditions, whether characterized as malignant,benign, hematologic, soft tissue or solid tumor. Specific cancersinclude digestive and gastrointestinal cancers, such as anal cancer,bile duct cancer, gastrointestinal carcinoid tumor, colon cancer,esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer,rectal cancer, appendix cancer, small intestine cancer and stomach(gastric) cancer. Neoplastic cells associated with either benign ormalignant tumors are associated with particular biomarkers that can beexploited as activators of an invention protide designed to be activatedin the particular context of the tumor.

One of the hallmarks of cancer as well as that of over seventy otherdiseases, including diabetic blindness, age-related maculardegeneration, rheumatoid arthritis and psoriasis, is the body's loss ofcontrol over angiogenesis. Angiogenesis-dependent diseases result whennew blood vessels either grow excessively or insufficiently. Excessiveangiogenesis occurs when diseased cells produce and release abnormalamounts of angiogenic growth factors, overwhelming the effects ofnatural angiogenesis inhibitors. The resulting new blood vessels feeddiseased tissues, which in turn destroy normal tissues. Therefore, abiological function of an effector incorporated within a protide of theinvention can be anti-angiogenic.

Upon their release, angiogenic growth factors diffuse into nearbytissues and bind to specific receptors located on the endothelial cellsof nearby preexisting blood vessels. Once growth factors bind to theirreceptors, the endothelial cells become activated and send signals fromthe cell surface to the nucleus. As a result, the endothelial cell'smachinery begins to produce new molecules including enzymes that createtiny holes in the basement membrane that surrounds existing bloodvessels. As the endothelial cells begin to proliferate, they migrate outthrough the enzyme-created holes of the existing blood vessel towardsthe diseased tissue; in the case of cancer, the endothelial cellsmigrate towards the tumor. Specialized molecules called adhesionmolecules, such as selectins or integrins, provide anchors that allowthe new blood vessel to sprout forward. Additional enzymes, among themmatrix metalloproteinases (MMPs), are produced to dissolve the tissue infront of the growing blood vessel tip to allow for its continued tissueinvasion. As the vessel extends, the tissue is remolded around thevessel and endothelial cells roll up to form a new blood vessel.Subsequently, individual blood vessels, connect to form blood vesselloops that can circulate blood. Finally, the newly formed blood vesselsare stabilized by specialized muscle cells (smooth muscle cells,pericytes) that provide structural support and blood flow through theneovascularized tissue begins. As those skilled in the art willappreciate, the enzymes described above as well as other art-knownenzymes associated with the progression of angiogenesis are useful asactivators associated with the context of angiogenesis.

Significantly, angiogenesis is one of the critical events required forcancer metastasis. Metastasis, the ability of cancer cells to penetrateinto lymphatic and blood vessels, circulate through the bloodstream, andinvade and grow in normal tissues elsewhere makes cancer alife-threatening disease. Tumor angiogenesis is the proliferation of anetwork of blood vessels that penetrates into cancerous growths,supplying nutrients and oxygen and removing waste products.

Therefore, the invention protides and related methods are characterized,in part, by their multifunctionality and context-specifity desired.Those skilled in the art will appreciate that, given the versatility ofeffectors and activators useful for practicing the invention asdescribed herein, a protide can be designed to initiate any two or morebiological functions in virtually any context desired.

In a further embodiment, the invention provides a method of treatinginfection by administering to a subject a therapeutically effectiveamount of the context-activated protide. The protide, also provided bythe invention, can have one activator site and two effector peptideshaving distinct biological functions, wherein the distinct biologicalfunctions are antimicrobial and immunomodulatory. One of the effectorpeptides in this embodiment can be interleukin-8, the other effectorpeptide can be defensin hNP-1, and the activator can be a protease suchas a clotting cascade protease or a complement fixing protease asdescribed herein. As shown in FIG. 4, antibody recognition of amicrobial pathogen, or the pathogen itself, can trigger classical oralternative complement cascades (respectively), resulting in relativelylocal production of C3 convertase serine proteases, either C4b2a orC3bBb. Each of these C3 convertase enzymes can serve as a protideactivator in this embodiment, cleaving the mosaic protide at thecleavage site, liberating the effector domains IL-8 and defensin hNP-1as independent molecules to effect their respective functions asdescribed above. Prior to and beyond the setting of activation in thecontext of the complement cascade, the mosaic construct remainscharge-neutralized and relatively inactive, minimizing the non-selectivetoxicity of hNP-1, and the indiscriminant stimulating effects of IL-8.Thus, complement fixation specifically upon pathogenic microorganismsand/or at sites of infection is the context that localizes and activatesthe protide.

In the specific embodiment depicted in FIG. 3, thrombin is the protideactivator that cleaves the mosaic protide at the cleavage site resultingin the liberation of the effector IL-8 and defensin hNP-1 antimicrobialpeptide domains into independent molecules to effect their respectivefunctions of unfavorable cytotoxic effects against host cells (hNP-1)and recruitment and stimulation of neutrophils to respond specificallyto the sites of infection (IL-8). Prior to thrombin-activation, themosaic construct is designed to be charge-neutralized and relativelyinactive, minimizing the non-selective toxicity of hNP-1, and theindiscriminant inflammation stimulating effects of IL-8. Thus, thrombinactivation at sites of vascular injury or infection itself localizes andactivates the protide.

In a further embodiment, the invention provides a method of treating aneoplastic condition by administering to a subject a therapeuticallyeffective amount of a of the invention, for example, a protideencompassing one activator site and two effector peptides havingdistinct biological functions, wherein the distinct biological functionscan include anti-neoplastic, pro-apoptotic, and anti-angiogeniceffectors. In a related embodiment, the invention provides a method oftreating a condition associated with decreased cell death byadministering to a subject a therapeutically effective amount of aprotide of the invention that has effectors with apoptotic andanti-angiogenic biological functions.

In a further embodiment, the invention provides a method of treating amicrobial infection by administering to a subject a therapeuticallyeffective amount of an antimicrobial protide of the invention containingsolely peptide effectors, also referred to an antimicrotide. The protideadministered in this embodiment of the invention can include two or moreeffectors that encompass, for example, an antimicrobial peptide,toxicity-neutralizing peptide, immunomodulatory peptide,ligand-targeting peptide, or any other polypeptide sequence expected tohave a specific function when activated in the context of microbialinfection or tissue injured due to infection.

In one embodiment, the invention provides a method of treating amicrobial infection by administering to a subject a therapeuticallyeffective amount of a hybrid protide of the invention, also termed anantibiotide. A protide can be introduced locally, regionally, orsystemically to treat an established infection, or a pathologicalcondition for which presumptive diagnosis indicates empiric therapy thatis consistent with eradicating or preventing the progression of anemerging or established infection. An activator of an invention protideuseful for treatment of a microbial infection can be associated witheither the microbe, for example, a microbial virulence factor or thehost organism, for example, a soluble response molecule or protein, or acell-associated compound.

An invention protide useful for treatment of a microbial infection canbe activated by a microbial virulence factor, exoenzyme or othersecreted product, surface component, unique metabolic process orpathway, target, or condition of the pathogen. Alternatively, a protidecan be activated by host responses to the pathogen. Examples of suchactivators include, for example, soluble components such as tissuefactor, thrombin, complement proteins, fragments of complementactivation, coagulation cascade components or reactants, clotdissolution components or reactants, and/or activated protein C orrelated agents. Also contemplated as activators useful for practicingthe invention are, cell-associated activators, for example, platelet,leukocyte, or lymphocyte products including surface-bound or releasedproteins, enzymes or other reactants.

In applications of the invention method involving an establishedinfection or a host response to infection, activators can be present orgenerated. An activator useful for activation of a protide of theinvention can be advantageously selected based on a high concentrationin the immediate proximity of the infection locus so as to allow foractivation of the majority of protides in the desired context. Oneskilled in the art will be able to select an activator that representsthe desired activation context. For applications of the inventionmethods in the arena of microbial infection, context-activation can bedesigned to specifically occur in the local context of infection so asto effect optimal relative protide effector concentrations in specificcontexts of infection. In addition to context activation that maximizesefficacy, the protides and methods of the invention also minimize thepotential for inadvertent host cytotoxicity in areas that do notrepresent the context. Therefore, in the absence of infection, theprotide activators are either absent or are present at concentrationsinsufficient for effective protide activation, thereby minimizinginadvertent or indiscriminant acute toxicity.

In addition to specific pathogen or host molecules that can serve asactivators as described above, protides can also be designed to becomeactivated to diagnose, prevent, or treat infection in unique and/orspecific biochemical or physiological contexts associated with microbialpathogens. Examples of such biochemical or environmental contextsinclude ionic, osmotic, pH, oxidation/reduction, or other conditionsthat are unique to, characteristic of, or present in the context ofinfection or disease processes that occur upon infection, or hostresponses to these events. For example, a protide can be designed torequire the influence of protonation, conformation change, or othermodification that occurs uniquely or disproportionately in the contextof acidic pH, to activate the protide or its ensuing effectors byaltering their structure-activity relationship(s) from inactive toactive. As one example, genitourinary tissues, such as renal tissues orgenitourinary mucosa, can exhibit pH values that are decreased normally,or in the setting of infection. A protide designed to be activated onlyunder such acidic conditions could be designed to either be vulnerableto activation in these conditions, or directly activated by theseconditions, and thus would be predicted to be active only in suchcontexts. Alternatively, protides can be designed to be inactive inparticular contexts or conditions, such as conditions of relatively highosmotic strength or relatively high pH, so as to minimize or preventuntoward or toxic effects such as nephro- or hepatotoxicity. By way of afurther example, activation as well as leukocyte accumulation areconditions associated with infection. Moreover, a fundamental strategyof host defense phagocytes is to phagocytize the microbial pathogen,subjecting it to the harsh environment of the acidic phagolysosome. Thecompartment so created can become acidified to pH values of 5.5 or loweras the leukocyte responds to the pathogen. Therefore, a protide can bedesigned that is activated or has amplified or antimicrobial activities,for example, by pH, phagolysosomal enzymes or reactants, or acombination of these conditions, or can amplify or potentiate theantimicrobial mechanisms of leukocytes or other host cells within suchsettings, so as to inhibit or kill pathogens that enter such cells.

Protide activation also can include conformational, oxidiation orreduction-mediated changes in disulfide array, assembly into multimersof two or more homomeric (identical) or heteromeric (non-identical)effectors, or other modifications of the protide and/or its subsequenteffectors. In a particular embodiment, protide activation is triggeredas a result of protide accumulation, or its resulting effectorcomponents, so as to achieve or surpass threshold concentrationsrequired to optimize or catalyze activation or activity throughmultimerization or other modification in structure or function of theprotide or its effectors.

It is understood, that activation can involve combinations of theprotide activation strategies described above. For example, a protidecan be designed that is not responsive to an activator unless both theprotide and the activator are present within a context associated withor resulting from infection or other disease.

Briefly, once a pathogen establishes infection and expresses requisitevirulence factor or other activators a protide of the invention can beintroduced that contains two functional effectors, for example, oneeffector that has direct activity against the pathogen and becomesamplified under specific conditions and a second effector that has achemokine-like motifs or functions. Upon protide activation in thepresence of the virulence factor activator in the context of infectionthe effector binds to and exerts activities against the pathogen thatare relatively modest and not toxic to the host. In addition, activationby the microbial virulence factor or in the greater context of infectionor disease results in diffusion away from the nidus of infection by thechemokine motif-containing effector, thereby creating a chemotacticgradient that results in influx of immunocompetent cells, for example,leukocytes. The chemokine motif-containing effector thus recruitsleukocytes to the site of infection where pathogens exposed to theantimicrobial effector are phagocytosed by the arriving leukocytes, andenter the acidic phagolysosome. The antimicrobial effector exertsamplified activity against the pathogen in the context of the acidicphagolysosome. In this embodiment, the antimicrobial effector functionsalong with the intrinsic antimicrobial mechanisms within the acidiccontext of the leukocyte phagolysosome, leading to destruction andclearance of the pathogen. The infection is resolved and the protide wasat no point activated beyond the context of infection, Since theantimicrobial effector exerts suboptimal activity under neutral oralkaline conditions of pH, toxicity to host cells and tissues isminimal. Alternatively, protides can be designed to prevent leukocyteactivation or accumulation in specific contexts where desirable,yielding for example an anti-inflammatory effect.

An activator can be selected based on the context desired foractivation. As an example, nearly all bacterial pathogens integratepeptidoglycan into their cell wall complex. Specific enzymes, includingcarboxypeptidases and transpeptidases, are necessary to activate andcrosslink peptidoglycan precursors to achieve the native matrix. Anantimcrobial protide of the invention can therefore be designed tocontain activator sites responsive to such activator enzymes thatinitiate or amplify an antimicrobial effector, as well as collateralinhibition of specific enzymes required for peptidoglycan synthesis. Anenzyme that is unique to a targeted context, for example, apeptidoglycan synthesis enzyme, which is unique to bacterial pathogens,is particularly useful as it can be activated in the particular localcontext of bacterial infection, thereby minimizing inadvertent host celltoxicity. Those skilled in the art will be able to select otheractivator enzymes that are specific to a desired context for protideactivation.

An antimicrobial protide of the invention can therefore be designed suchthat is activated in a broad context, for example, by selecting anactivator that is common to a variety of pathogens or a variety ofcancers. Alternatively, a more narrow context can be selected bypreparing a protide that encompasses one or more activator sites thatare triggered by activators particular to such a narrower context. Ifdesired, a protide of the invention can be designed so as to beactivated in the context of Gram-positive versus Gram-negative bacteria.Gram-positive bacteria utilize so-called LPXTG(Leucine-Proline-X-Threonine-Glycine) (SEQ ID NO: 5) or related motifsin structural proteins intended for cell wall or extracellular membranesurface localization. Numerous surface proteins integral for virulenceof Gram-positive pathogens such as S. aureus and S. pyogenes areanchored to the Gram-positive cell wall/envelope complex via a proteinprocessing mechanism, utilizing a C-terminal sorting sequence with anLPXTG (SEQ ID NO: 5) motif. Sortase enzymes are membrane proteins commonto many Gram-positive and Gram-negative pathogens that cleave precursorproteins intended for the cell wall/envelope complex between threonineand glycine residues found within the LPXTG (SEQ ID NO: 5) motif. Thus,as an example, a given sortase can catalyze the formation of a covalentamide bond between the carboxyl-sidegroup of threonine andamino-sidegroup of adjacent peptidoglycan. Significantly, the sortasemechanism is predominant in several Gram-positive pathogens including S.aureus and S. epidermidis, streptococci such as S. pyogenes, S.pneumoniae, and S. agalactiae, enterococci such as E. faecium and E.faecalis, as well as difficult Gram-negative pathogens such as Ps.aeruginosa and various members of the family Enterobacteriaceae, whereit is integral for maturation of surface protein attachment to the cellwall/envelope complex. Protides can therefore be prepared that containLPXTG (SEQ ID NO: 5) activation sites that allow exploitation of thesortase mechanism to liberate one or more antimicrobial effectors,leading to competitive inhibition of sortase, which can also beoutcompeted by the activator sites from acting on natural substrates tocatalyze essential surface protein anchoring.

A further application of an invention protide with antimicrobialbiological function is in a species-specific context. Individual speciesor specific strains of microbial pathogens account for a largeproportion of infections including infections due to strains thatelaborate penicillinase or cephalosporinase enzymes such as S. aureus orPseudomonas aeruginosa (P. aeruginosa); pathogens that express highlevels of one or more exoenzymes that are believed crucial to virulencesuch as coagulase of S. aureus; a variety of serine proteases elaboratedby Gram-negative pathogens such as DegS or DegQ in Escherichia coli andAlgW or MucD in P. aeruginosa; and phospholipases or aspartyl proteasesof the yeast Candida albicans (C. albicans). Each of the aforementionedvirulence factors provides an example of an activator that correspondsto an activator site of an invention protide. Thus, an invention protidecan be prepared to be activated by, for example, one or more virulencefactors, enzymes, or specific targets of a microbial pathogen as well asany combination thereof. For example, a protide containing D-ala/D-alamotif within its activator site can be designed to mimic a peptidoglycanprecursor, such that it becomes activated by peptidoglycan enzymes,thereby liberating antimicrobial peptides in close proximity to thevulnerable cell membrane of the pathogen. Alternatively, examples ofprotides can also be envisioned in which the activator sites arespecific substrates for one or more exoenzymes or other virulencefactors required for infection by a pathogen.

In a further embodiment, the invention provides a method of preventingthe establishment of infection or onset of symptoms due to the presenceof the pathogen in normal individuals, or those at increased risk ofinfection for any reason. Settings or conditions associated withincreased of infection that are appropriate for practicing the inventionmethods for prophylactic use of protides include, for example, acute orchronic immunosuppression associated with organ transplantation; chronicsteroids use in allergy, autoimmune, rheumatoid, or other pathologicalconditions; diabetes or other pathological conditions attributed toreduced immune functions; cancer; cancer chemotherapy; individualsrequiring long-term vascular access, such as those undergoing kidneydialysis or that need ongoing vascular catheterization; infectionresulting in an immunocompromised state such as HIV; pre- orpost-surgical procedures, or other settings or pathological conditionsknown to those skilled in the art. In such settings, a protide can beused as a prospective prophylactic agent or pre-antibiotic to protectagainst pathogens should they colonize and elaborate virulence factor orhost response activators directly or indirectly, or that modify aphysiological context such that a protide is activated. For example, aprotide can be administered to a patient at risk of infection, but notactivated unless the pathogens are or become present and virulent, forexample, expressing virulence factor activators, or become opportunisticvia an increase in pathogen quantity via proliferation, or theirupregulation of virulence factor expression to cause overt infection.Absent these conditions, the protide remains inactive and relativelyharmless to the host.

Similarly, the context in which an antineotide is activated can bechosen by the user designing the protide. If a broad context is desiredfor actication, for example, in the context of any tumor cell type, anactivator can be chosen that ubiquitous to tumor cells, for example, ametalloproteinase. Alternatively, if activation in a more narrow contextis desired, an activator can be selected that represents a particularorgan or cell type, for example, Prostate Specific Antigen (PSA) orTMPRSS2, which are serine proteases that are overexpressed in a majorityof prostate cancer patients. PSA has been shown to directly degradeextracellular matrix glycoproteins such that any of the degradedproducts also are localized in the context of a protate neoplasticcondition and also can be useful as context-specific activators for aninvention protide.

An individual undergoing cancer chemotherapy has a significantlyincreased risk of infection and represents an example of an appropriatetarget for prophylactic application of the invention protides andmethods. A protide can be introduced prior to and throughout the periodof time during which immune suppression is acute as a result ofchemotherapy. For example, it is known that a time of great risk forinfection often flanks the pre- and post-nidus leukocyte count interval(eg., neutropenia). Hallmark pathogens often associated with suchcontexts include fungal pathogens that are very difficult to treat onceinfection has been established such as, for example, Candida,Aspergillus and Mucor, as well as bacterial pathogens that can overwhelman immunocompromised host such as, for example, Staphylocococcus,Pseudomonas and Enterobacteriaceae. Given the teachings provided herein,the skilled person will appreciate that protides or appropriatecombinations of protides can be specifically used as prophylactic agentsagainst pathogens common to a respective risk of infection. As describedabove, unless activated, the protide in its inactive form would beessentially inert or exert reduced activity or cytotoxicity, and clearedfrom the system with little or no adverse effect. However, should theindividual become colonized with or infected by a relevant pathogen, theprotide(s) becomes exposed to activators, either from the organism orfrom host responses to the organism such as the organisms virulencefactor or exoenzyme, or a host-generated factor that is specific to thedesired context because of tissue damage or other effects of infection.In such a prophylactic environment, the effector's biologicalfunction(s) that combat the organism can be initiated prior to or at theearliest point in the establishment of infection and can be locallyconcentrated to the specific context in which activators are present insufficient quantity or quality, and/or in a milieu consistent withactivation.

In a further embodiment, the present invention provides protides andrelated methods for reconstituting normal homeostasis in individualswith deficiencies in genes or gene products. For example, a protide ofthe invention can be prepared to be activated in the absence of aprotein or other factor associated with normal function. In thisembodiment, a protide can be designed that, if exposed to an abnormalcondition caused by, for example, the absence of a particular protein,it is subject to degradation by an activator, yielding the deficientprotein as an effector, thereby reconstituting the normal condition.Such a protide can be used to reconstitute normal homeostasis inindividuals having inherited or acquired defects or deficiencies in oneor more gene products.

In a further embodiment, a protide can be prepared that is activatedonly in the presence of a microorganism, tumor cell, other pathogeniccell, or a component, exoproduct, or metabolite of such, so that theeffectors are detectable directly or indirectly for diagnostic purposes.In such a diagnostic embodiment, an effector can be appropriately chosento allow for easy detection through, for example, calorimetric,immunologic, electronic, radiographic, biochemical or other techniqueknown to those skilled in the art. Thus, in a diagnostic application ofthe invention protides and methods, specific protides can be preparedthat detect and/or diagnose a specific disease or pathogenic state, ortheir etiologies.

As described herein, an invention protide can have a cascade regulatingfunction (FIG. 11). As described herein, such a protide also referred toas cascatide can be designed to amplify or inhibit an endogenouscascade, for example, a signal transduction cascade or coagulationcascade. In a further embodiment related to cascade-regulating protidesor cascatides, the invention provides both pro-apoptotic andanti-apoptotic protides. In particular, a protide can be designed andprepared with one or more activator sites that are specific substratesfor one or more specific caspase enzymes, which are expressed when theprocess of apoptosis is initiated and serve as activators. In thisembodiment, the protide is activated to liberate its effector(s) onlywhen apoptosis is activated, at which time the released effector(s)target mitochondria, for example, by means of specific sequence,structure, or composition of the effector, and either amplify theinitial apoptotic signal or inhibit the response. If desired, aneffector can also be an inhibitor or activator of a particular caspase.A pro-apoptotic cascatide can be useful in applications directed atreducing the severity of a condition associated with a reduction ordeceleration in cell, for example, cancer. An anti-apoptotic cascatidecan be useful in applications directed at reducing the severity of acondition associated with increased cell death, for example, in thetreatment of an ischemic condition, neurodegenerative condition,autoimmune condition, or in anti-aging therapy.

A protide of the invention useful for practicing the methods of theinvention can be formulated and administered by those skilled in the artin a manner and in an amount appropriate for the pathological conditionto be treated, for example, an infection, neoplastic disorder,inflammation; the rate or amount of inflammation; the weight, gender,age and health of the individual; the biochemical nature, bioactivity,bioavailability and side effects of the particular compound; and in amanner compatible with concurrent treatment regimens. An appropriateamount and formulation for decreasing the severity of a pathologicalcondition in humans can be extrapolated from credible animal modelsknown in the art of the particular disorder. It is understood, that thedosage of a therapeutic substance has to be adjusted based on thebinding affinity of the substance, such that a lower dose of a substanceexhibiting significantly higher binding affinity can be administeredcompared to the dosage necessary for a substance with lower bindingaffinity. For an invention protide several factors can be taken intoaccount when determining the proper dosage, for example, the nature ofthe protide effectors and their bioactivity upon activation, theanticipated concentration of activator and the responsiveness of theactivator site to presence of the activator.

The total amount of protide can be administered as a single dose or byinfusion over a relatively short period of time, or can be administeredin multiple doses administered over a more prolonged period of time.Such considerations will depend on a variety of case-specific factorssuch as, for example, whether the disease category is characterized byacute episodes or gradual or chronic deterioration. For an individualaffected with an acute infection or inflammatory response, for example,as associated with a bacterial infection, the substance can beadministered as a single dose or by infusion of several large doses in arelatively short period of time. For an individual affected with chronicdeterioration, for example, as associated with a neuroinflammatorydisorder, the substance can be administered in a slow-release matrice,which can be implanted for systemic delivery or at the site of thetarget tissue, which means an area proximal to the desired context.Contemplated matrices useful for controlled release of therapeuticcompounds are well known in the art, and include materials such asDepoFoam™, biopolymers, micropumps, and the like.

The protides administered in the methods of the invention can beadministered to the individual by any number of routes known in the artincluding, for example, systemically, such as intravenously orintraarterially. A therapeutic protide can be provided in the form ofisolated and substantially purified polypetides in pharmaceuticallyacceptable formulations using formulation methods known to those ofordinary skill in the art. These formulations can be administered bystandard routes, including for example, topical, transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,intravaginal, intrauterine, oral, rectal or parenteral such asintravenous, intraspinal, intrathecal, subcutaneous or intramuscularroutes. Intrathecal administration of a therapeutic protide into theintradural or subarachnoid space can be an appropriate route fordecreasing the severity of a neuroinflammatory condition. Intravenousadministration of a terhapeutic substance containing a protide also is apreferred route for practicing the invention. In addition, a therapeuticsubstance administered in the methods of the invention can beincorporated into biodegradable polymers allowing for sustained releaseof the substance useful for prophylactic and reconstitutive applicationsdescribed above. Biodegradable polymers and their use are described, forexample, in Brem et al., J. Neurosurg. 74:441-446 (1991), which isincorporated herein by reference.

The methods for treating a particular pathological conditionadditionally can be practiced in conjunction with other therapies. Forexample, for treating cancer, the methods of the invention can bepracticed prior to, during, or subsequent to conventional cancertreatments such as surgery, chemotherapy, including administration ofcytokines and growth factors, radiation or other methods known in theart. Similarly, for treating pathological conditions which includeinfectious disease, the methods of the invention can be practiced priorto, during, or subsequent to conventional treatments, such as antibioticadministration, against infectious agents or other methods known in theart. Treatment of pathological conditions of autoimmune disorders alsocan be accomplished by combining the methods of the invention forinducing an immune response with conventional treatments for theparticular autoimmune diseases. Conventional treatments include, forexample, chemotherapy, steroid therapy, insulin and other growth factorand cytokine therapy, passive immunity and inhibitors of T cell receptorbinding. The protides of the invention can be administered inconjunction with these or other methods known in the art and at varioustimes prior, during or subsequent to initiation of conventionaltreatments. For a description of treatments for pathological conditionscharacterized by aberrant cell growth see, for example, The MerckManual, Sixteenth Ed, (Berkow, R., Editor) Rahway, N.J., 1992.

As described above, administration of an invention protide can be, forexample, simultaneous with or delivered in alternative administrationswith the conventional therapy, including multiple administrations.Simultaneous administration can be, for example, together in the sameformulation or in different formulations delivered at about the sametime or immediately in sequence. Alternating administrations can be, forexample, delivering a protide formulation and a conventional therapeutictreatment in temporally separate administrations. Temporally separateadministrations of a compound, immunomodulatory flagellin peptide,polypeptide or modification thereof, and conventional therapy can usedifferent modes of delivery and routes.

A therapeutic protide-containing substance administered in the methodsof the invention also can be administered as a solution or suspensiontogether with a pharmaceutically acceptable medium. Such apharmaceutically acceptable medium can include, for example, sterileaqueous solvents such as sodium phosphate buffer, phosphate bufferedsaline, normal saline or Ringer's solution or other physiologicallybuffered saline, or other solvent or vehicle such as a glycol, glycerol,an oil such as olive oil or an injectable organic ester. Apharmaceutically acceptable medium can additionally containphysiologically acceptable compounds that act, for example, stabilizethe neutralizing agent, increase its solubility, or increase itsabsorption. Such physiologically acceptable compounds include, forexample, carbohydrates such as glucose, sucrose or dextrans;antioxidants such as ascorbic acid or glutathione; receptor mediatedpermeabilizers, which can be used to increase permeability of theblood-brain barrier; chelating agents such as EDTA, which disruptsmicrobial membranes; divalent metal ions such as calcium or magnesium;low molecular weight proteins; lipids or liposomes; or other stabilizersor excipients. Those skilled in the art understand that the choice of apharmaceutically acceptable carrier depends on the route ofadministration of the compound containing the protides and on itsparticular physical and chemical characteristics.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions such as the pharmaceuticallyacceptable mediums described above. The solutions can additionallycontain, for example, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient. Otherformulations include, for example, aqueous and non-aqueous sterilesuspensions which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example, sealed ampules and vials, and can be stored in alyophilized condition requiring, for example, the addition of thesterile liquid carrier, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

For applications that require the protide-containing compounds andcompositions to cross the blood-brain barrier, formulations thatincrease the lipophilicity of the compound are particularly desirable.For example, the neutralizing agent can be incorporated into liposomes(Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press,Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids orother lipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

A therapeutic protide-containing substance administered in the methodsof the invention can also be prepared as nanoparticles. Adsorbingpeptide compounds onto the surface of nanoparticles has proven effectivein delivering peptide drugs to the brain (see Kreuter et al.; BrainResearch 674:171-174 (1995)). Exemplary nanoparticles are colloidalpolymer particles of poly-butylcyanoacrylate with a therapeuticprotide-containing substance to be administered in the methods of theinvention adsorbed onto the surface and then coated with polysorbate 80.

Image-guided ultrasound delivery of a therapeutic protide-containingsubstance administered in the methods of the invention through theblood-brain barrier to selected locations in the brain can be utilizedas described in U.S. Pat. No. 5,752,515. Briefly, to deliver atherapeutic substance past the blood-brain barrier a selected locationin the brain is targeted and ultrasound used to induce a changedetectable by imaging in the central nervous system (CNS) tissues and/orfluids at that location. At least a portion of the brain in the vicinityof the selected location is imaged, for example, via magnetic resonanceimaging (MRI), to confirm the location of the change. An therapeuticsubstance administered in the methods of the invention into thepatient's bloodstream can be delivered to the confirmed location byapplying ultrasound to effect opening of the blood-brain barrier at thatlocation and, thereby, to induce uptake of the substance.

In addition, polypeptides called receptor mediated permeabilizers (RMP)can be used to increase the permeability of the blood-brain barrier tomolecules such as therapeutic, prophylactic or diagnostic substances asdescribed in U.S. Pat. Nos. 5,268,164; 5,506,206; and 5,686,416. Thesereceptor mediated permeabilizers can be intravenously co-administered toa host with molecules whose desired destination is the cerebrospinalfluid compartment of the brain, for example, in the treatment of aneuroinflammatory condition. The permeabilizer polypeptides orconformational analogues thereof allow therapeutic substances topenetrate the blood-brain barrier and arrive at their target destinationwhich can be selected based on its proximity to the desired activationcontext. Such polypeptides can be designed as part of strategicinvention protides.

In current treatment regimes for most diseases, more than one compoundis often administered to an individual for management of the same ordifferent aspects of the disease. Similarly, in the methods of theinvention for treating a vascular injury, neoplastic condition,microbial infection, a condition associated with decreased cell death orinflammatory condition, a therapeutic protide-containing substance canadvantageously be formulated with a second therapeutic compound such asan anti-inflammatory compound, antimicrobail compound, chemotherapeuticcompound, immunosuppressive compound or any other compound that managesthe same or different aspects of the particular disease. As an example,for treatment of an infectious disease a therapeutic substance canadvantageously be formulated with a second therapeutic compound such asan antibiotic. Contemplated methods of treating a pathological conditionby administering to a subject a therapeutically effective amount of aninvention protide therefore include administering a therapeuticsubstance useful in the methods of the invention alone, in combinationwith, or in sequence with, such other compounds. Alternatively,combination therapies can consist of fusion proteins, where atherapeutic substance useful for treating a particular pathologicalcondition is linked to a heterologous protein, such as an inventionprotide. It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Design and Functional Confirmation of Protide-1

This example describes the design of Protide-1 (PT-1), a peptideantibiotide with distinct effector and activator domains. The examplefurther describes the confirmation of activity of PT-1.

PT-1 consists of two effectors and one activator site. In particular,PT-1 contains an antimicrobial peptide effector (RP-1), a chemokine-likepeptide effector (IL-8 domain), and an activator site specific forstaphylococcal V8 protease, one of numerous virulence factors that iselaborated by S. aureus in order to establish and proliferate infection.PT-1 was designed to be cleaved into two distinct effectors in thepresence of the activator, staphylococcal V8 protease. In particular,PT-1 was designed to exert antimicrobial activity less than that of theantimicrobial peptide RP-1 in the absence of V8 protease, butantimicrobial activity equivalent to or exceeding that of RP-1 in thepresence of V8 protease produced by S. aureus. Thus, PT-1 was designedto exert optimal antimicrobial activity in the context of infections dueto staphylococcal cells elaborating the virulence factor V8 protease.

Since PT-1 is a peptide antibiotide, it was synthesized entirely bysolid-phase peptide synthesis via conventional F-moc synthetic chemistrywell known in the art and described in detail elsewhere. PT-1 wassynthesized in the Biopolymer Core Facility, Harbor-UCLA REI utilizing aSymphony Multiplex peptide synthesizer and Rainin Instruments. Followingpeptide chain assembly, the complete PT-1 peptide was cleaved from theresin, precipitated, and washed per standard techniques. Preparativereverse-phase HPLC (RP-HPLC) employing water/acetonitrile gradientscontaining 0.01% trifluoroacetic acid (TFA) yielded subsequentpurification of PT-1, which typically achieved >85% baseline purity.Purified PT-1 was subsequently assessed by analytical RP-HPLC andacid-urea polyacrylamide gel electrophoresis (AU-PAGE) to ensure purity,amino acid analysis and modified Lowry assay to verify quantity, andMALDI-TOF mass spectrometry to corroborate correct synthesis via mass.

Similarly, the antimicrobial peptide RP-1 was synthesized, purified, andverified for purity and correct mass substantially as described ininternational patent publication WO99/429119, which is incorporatedherein by reference in its entirety.

In order to assess the antimicrobial properties of PT-1, a panel ofwell-characterized test pathogens was used that included S. aureusstrains ISP479C and ISP479R as described by Dhawan et al., Infect.Immun. 66:3576-3479 (1998); Candida albicans strains 36082 (ATCC typestrain 36082)and 36082R as described by Yeaman et al., Infect. Immun.64:1379-1384 (1996); Salmonella typhimurium strains m5996s and 14028s asdescribed by Fields et al., Science 243:1059-62 (1989); Yeaman et al.,38th Interscience Conference on Antimicrobial Agents and Chemotherapy(ICAAC), San Diego, Calif., Abstract No. F-170 (1999); and Bacillussubtilis strain ATCC 6633 as described by Yeaman et al., Infect. Immun.60:1202-1209 (1992), each of which is incorporated herein by referencein its entirety.

In order to compare the antimicrobial activities of PT-1 with those ofRP-1 against a panel of test pathogens, radial diffusion assay and amicrotiter well assay were utilized.

Briefly, a modified radial diffusion assay was employed to compare theantimicrobial activities of PT-1 with RP-1 in a solid-phase matrixformat. In this assay, organisms were introduced at an inoculum of 10⁶CFU/ml into a solution of 1% (wt/vol) molecular grade agarose andappropriate levels of mM glucose in PIPES buffer (PH 5.5 or 7.5; meltedand then cooled to 42° C. prior to introduction of the organisms). Theliquid underlayer matrix containing test organism was then vortexed,poured into a petri dish, and allowed to solidify. Wells (4 mm) wereremoved from the solidified underlayer, into which 10 ul samples of PT-1or RP-1 were added to achieve a final concentration of 5 ug per well.The reaction plates were subsequently incubated at 37° C. for 3 h,followed by overlay with nutrient or Sabaroud's agar as appropriate forbacterial or fungal test pathogens. The assay plates were then incubatedfor 18 h at 37° C., after which zones of inhibition (in mm of diameter)were measured and recorded.

A microtiter well assay was also performed as a complement to the radialdiffusion assay described above. In this assay, PT-1, RP-1, or V8protease (Sigma Chemical Co.) were serially diluted from 100 ug (range,100-0.20 ug/ml) and individually assessed for antimicrobial activityagainst S. aureus strains ISP479C and ISP479R as described above. Inaddition, this assay allowed for the evaluation of antimicrobialactivities of combinations of PT-1 and V8, in conditions of constant ordynamic dilution of one or both agents. This latter approach enabled thedetermination of static as well as dynamic fractional inhibitoryconcentrations (FICs) of PT-1 in the presence and absence of purified V8protease.

When exposed to purified V8 protease in vitro, PT-1 was in a time frameof minutes cleaved (activated) into two separate fragments that weredistinguishable upon analytical RP-HPLC as well as acid-ureapolyacrylamide gel electrophoresis. These fragments exhibited retentiontimes that were consistent with those of RP-1 and IL-8-like domains thatcompose the pre-cleaved form of PT-1.

In radial diffusion assays, PT-1 demonstrated substantially lessantimicrobial activity than RP-1 against all organisms except S. aureus.Therefore, an absence of V8 protease in organisms other than S. aureusresulted in no activation of PT-1, consistent with the concept anddesign of PT-1, since only S. aureus elaborates V8 protease. However,activation and enhanced antimicrobial activity of PT-1 was observed forboth S. aureus strains, regardless of their intrinsic susceptibility orresistance to related antimicrobial agents. This demonstrates that thePT-1 protide is context-activated.

In microtiter assays, PT-1 in the presence of purified V8 proteaseconsistently demonstrated a 2-fold or more increased activity comparedto PT-1 in the absence of this activator. For example, when co-incubatedwith 1 ml V8 protease, PT-1 exerted an MIC against S. aureus strains of3.1 ug/ml, as compared with its MIC of 12.5 without V8 protease. Thesefindings convey an FIC of <0.5, considered to represent a synergisticrelationship between PT-1 activation by V8 protease, and enhancedactivity of the resulting effector against the test strain.

The above results exemplify the design and functional confirmation of aprotide by demonstrating that PT-1 has enhanced antimicrobial activityin the context of V8 protease.

Throughout this application various publications have been referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A context-activated protide comprising oneactivator site and two effector peptides having distinct biologicalfunctions, wherein said distinct biological functions are antimicrobialand immunomodulatory, wherein said context-activation results from theassociation between an enzyme expressed by a bacterial pathogen withsaid activator site, and wherein said immunomodulatory functioncomprises mediation of an immune response that is specific for a targetantigen or mechanism of pathogenesis.
 2. The context-activated protideof claim 1, wherein said context-activation initiates said biologicalfunctions of said effectors.
 3. The context-activated protide of claim1, wherein said bacterial pathogen is S. aureus.
 4. Thecontext-activated protide of claim 1, wherein said S. aureus belongs toa methicillin-resistant strain.
 5. The context-activated protide ofclaim 3, wherein said S. aureus is selected from the group consisting ofa vancomycin-resistant strain and intermediate-resistant to vancomycinstrain.
 6. The context-activated protide of claim 3, wherein said enzymeis a surface protein transpeptidase (sortase).
 7. The protide of claim1, wherein said antimicrobial function comprises directly killing saidbacterial pathogen.
 8. The protide of claim 1, wherein saidantimicrobial function comprises inhibition of said surface proteintranspeptidase (sortase).
 9. A context-activated protide comprising oneactivator site and two effector peptides having distinct biologicalfunctions, wherein said distinct biological functions are antimicrobialand immunomodulatory, and wherein said context-activation results fromthe association between an enzyme expressed by a bacterial pathogen withsaid activator site, and wherein said immunomodulatory functioncomprises trafficking cytokine functional groups or immune effector celltypes to a site of inflammation.
 10. The protide of claim 9, wherein oneof said effector peptides comprises interleukin-8, and wherein saidother effector peptide comprises Defensin hNP-1, and wherein saidactivator comprises a protease.